The invention relates to the use of peptides individually or in combination, for treating and/or preventing angiogenesis. It also relates to the use of a combination of peptides referred to as MuJ-7 as an anticancer drug in restricting tumor growth and its spread by inhibiting tumor angiogenesis. MuJ-7, in addition inhibits metastasis through its antiangiogenic activity in all cancers. The invention also relates to a pharmaceutical composition containing either individual peptides or combinations of peptides, and methods of treatment of human beings and animals for curing and/or preventing angiogenesis.
Angiogenesis is the growth of new microvessels. This process depends mainly on locomotion, proliferation, and tube formation by capillary endothelial cells. During angiogenesis, endothelial cells emerge from their quiescent state and can proliferate as rapidly as bone marrow cells, but unlike the bone marrow, angiogenesis is usually focal and of brief duration. Pathologic angiogenesis, while still a focal process, persists for months or years. The angiogenesis that occurs in diseases of ocular neovascularisation, arthritis, skin diseases, and tumors rarely terminates spontaneously and has until recently, been difficult to suppress therapeutically. Therefore, the fundamental goal of all antiangiogenic therapy is to return foci of proliferating microvessels to their normal resting state, and to prevent their regrowth1.
Although the molecular mechanisms responsible for transition of a cell to angiogenic phenotype are not known, the sequence of events leading to the formation of new vessels has been well documented2,3. The vascular growth entails either endothelial sprouting3,4 or intussusception5. In the first pathway, the following sequence of events may occur: (a) dissolution of the basement of the vessel, usually a postcapillary venule, and the interstitial matrix; (b) migration of endothelial cells toward the stimulus; (c) proliferation of endothelial cells trailing behind the leading endothelial cell(s); (d) formation of lumen (canalization) in the cndothelial array/sprout; (e) formation of branches and loops by confluence/anastomoses of sprouts to permit blood flow; (f) investment of the vessel with pericytes; and (g) formation of basement membrane around the immature vessel2,3. New vessels can also be formed via the second pathway: insertion of interstitial tissue columns into the lumen of preexisting vessels. The subsequent growth of these columns and their stabilization result in partitioning of the vessel lumen and remodelling of the local vascular network5,6.
The rationale for antiangiogenic therapy is that progressive tumor growth is angiogenesis-dependent8. The switch to the angiogenic phenotype appears to be an independent event that occurs during the multistage progression to neoplasia9. The angiogenic switch itself, while relatively sudden and well localized, is nonetheless a complex process. This phenotype is currently understood in terms of a shift in the net balance of stimulators and inhibitors of angiogenesis, during which inhibitors are down regulated10,11.
Once new capillary loops converge toward a small in situ carcinoma or a microscopic metastasis, the tumor cells are bathed in additional survival factors and growth factors, not only from the circulating blood (perfusion effect) but also from vascular endothelial cells themselves (paracrine effect). The positive regulators of angiogenesis include at least 14 angiogenic proteins that have been discovered during the past 12 years and which have been sequenced and cloned12. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are the most well studied and are found in a majority of different types of human tumors. During the angiogenic switch one or more of these angiogenic stimulators is upregulated and it appears, however, that this up regulation of angiogenic stimulators is accompanied by down regulation of local tissue inhibitors of angiogenesis.
The paracrine stimulation of tumor cells by products from endothelial cells also operates in the other direction. Endothelial cell survival and growth are driven by tumor derived mitogens and motogens. These findings have led to a model of tumor growth in which the endothelial cell compartment and the tumor cell compartment interact with each other. They not only stimulate each other""s growth, but if the endothelial cells are made unresponsive to angiogenic stimuli from the tumor cells, by administration of a specific endothelial inhibitor, both primary tumors11 and metastatic tumors10 can be held dormant, at a microscopic size. One could take advantage of this difference between endothelial cells and tumor cells by administering an angiogenesis inhibitor together with conventional cytotoxic chemotherapy up to the point at which the cytotoxic therapy would normally be discontinued because of toxicity or drug resistance. The angiogenesis inhibitor(s) could then be continued (for years), to maintain either stable disease or tumor dormancy1. Such combinations of antiangiogenic and cytotoxic therapy in tumor-bearing animals have been curative, whereas either agent alone is merely as inhibitor15.
Peptides/Proteins have previously been studied for antiangiogenic activity. Thrombospondin-1 (TSP-1) which is a naturally occurring inhibitor of angiogenesis, makes endothelial cells unresponsive to a wide variety of inducers. Both native TSP-1 and small antiangiogenic peptides derived from it show that this inhibition is mediated by CD3616 (Dawson et al., 1997). Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells.
The family of tissue inhibitors of metalloproteinases (TIMPs) are known to be specific inhibitors of matrix metalloproteinases (MMPs). The local balance between MMPs and TIMPs is believed to play a major role in extracellular matrix (ECM) remodelling during diseases such as cancer. TIMP-3 which is unique in being a component of ECM, inhibits endothelial cell migration and tube formation in response to angiogenic factors17 (Anand-Apte et al., 1996).
The conditioned medium of human promyelocytic leukemia (HL6O) cells has been shown to contain a cell growth inhibitory factor, human cytostatin. Human cytostatin can inhibit endothelial cell proliferation, migration and microvessel tube formation on Matrigel-coated surfaces18 (Yeung AK et al., 1996). Furthermore the anti-angiogenic effect of human cytostatin has been demonstrated on the chick chorioallantoic membrane. Human cytostatin can inhibit new blood vessel development, but cannot regress existing blood vessels.
Angiostatin, which is a 38 kD internal fragment of plasminogen is an antiangiogenic endothelial cell inhibitor and suppresses the growth of primary Lewis lung carcinoma in vivo19 (Wu, Z. et al., 1997).
Thalidomide has recently been shown to antagonize basic fibroblast growth factor-induced angiogenesis in the rat corneal micropocket assay. It has been suggested that thalidomide elevates tumor hypoxia in the Lewis lung tumor, presumably via an antiangiogenic mechanism20 (Minchinton AI et al., 1996).
One study examined the in vitro antiangiogeneic effects of the somatostatin analog octreotide on the growth of human umbelical vein endothelial cells (HUVEC) and vascular cells from explants of rat aorta cultured on fibronectin-coated dishes or included in fibrin gel. A total 10xe2x88x929M octreotide reduced the mean uptake of 3H-thymidine by HUVEC cells by 37% compared with controls. The 10xe2x88x928M concentration of octreotide inhibited the proliferation of endothelial and smooth muscle cells growing on fibronectin by 32.6% and reduced the sprouting of cells from the adventitia of aortic rings in fibrin by 33.2% compared with controls, as measured by tetrazolium bioreduction and image analysis, respectively. These results demonstrate that octreotide is an effective inhibitor of vascular cell proliferation in vitro21 (Danesi R. Del Taccam, Metabolism August 1996. 45(8 Supp 1: 49-50). In another experiment, somatostatin analogs SMS 201-995 and RC-160 were found to inhibit angiogenesis in the chick chorioallantoic membrane of the developing chicken embryo. Somatostatin analogues were associated in a dose-related fashion with both a greater percentage of inhibition of blood vessel growth and an increased grade of inhibition. It was hypothesized that inhibition of angiogenesis may be a mechanism responsible for the tumor regression observed in vivo following SMS or RC-160 therapy22 (Woltering E A et al., J. Surg. Res. March 1991, 50(3): 245-251).
The proinflammatory neuropeptide, substance P, stimulated angio-genesis in an in vitro model using HUVECs cultured on a basement membrane (Matrigel) substrate. Substance P stimulated endothelial cell differentiation into capillary-like structures in a dose-dependent manner. Stimulation of endothelial cell differentiation is a newly recognized biological function of substance P23 (Wiedermann, C. J. et al. Eur. J. Pharmacol. Mar. 18, 1996, 298 (3): 335-338).
The effects of Nitric oxide (NO) generation and endogenous production of NO elicited by substance P (SP) in angiogenic process were evaluated in the rabbit cornea (in vivo) and by measuring growth and migration of endothelial cells (in vitro). The NO synthase inhibitors given systemically inhibited angiogenesis elicited by [sar9]-SP-Sulfone. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors. Exposure of the cells to SP activated the calcium-dependent NO synthase. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis24 (Ziche M. et al., J. Clin. Invest. November 1994, 94(5):2036-2044).
The angiogenic activity of four vasoactive peptides with a range of vasodilator and vasoconstrictor properties were investigated in a rat sponge model. 2 daily doses of vasodilator peptide VIP(10 pmol) when given with interleukin-1 alpha caused intense neovascularization which was inhibited by simultaneous administration of VIP(10-28), a specific VIP receptor antagonist. These data show that VIP possesses angiogenic activity and the blockade of VIP induced angiogenesis at the receptor level could provide a strategy for the management of angiogenic disease25.
Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor alpha, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin adherent cells than in suspended cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression (Short S M et al, Molecular Biology of the Cell 9(8):1969-1980, 1998).
Cell adhesion to the extracellular matrix (ECM) has been implicated in apoptosis in anchorage-dependent cell types. It was recently found that a peptide derived from fibronectin (termed III14-2) inhibits the integrin-mediated cell adhesion to ECM. Using this antiadhesive peptide and a variety of ECM proteins, a critical role of the integrin-ECM protein interaction in apoptotic regulation of human umbilical vein endothelial cells (HUVEC) has been demonstrated. HUVEC in suspension undergoes apoptosis under the serum-free conditions, as judged by nuclear and DNA fragmentations (Fukai F et al, Experimental Cell Research, 242(l):92-99, 1998).
It has been previously shown that vasoactive intestinal polypeptide (VIP) induces endothelium-dependent relaxation of the human uterine artery. Non-competitive antagonism with methylene blue revealed that the pKa value for VIP-receptor complex was 8.10+/xe2x88x920.10 (n=6) and the receptor reserve expressed as K-A/EC50 was 0.89+/xe2x88x920.11, where pKa=log (10)K(A), and K-A is the dissociation constant of VIP-receptor complex (Jovanovic A. et al. Molecular Human Reproduction, 4(1):71-76, 1998).
Somatostatin (SRIF) exerts antiproliferative effects, and has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. 3 SRIF (0.1-1000 nM) caused a concentration-dependent inhibition of the bFGF-stimulated regrowth in CHO-K1 cells expressing human sst(2) (h sst(2)) or sst(5) (h sst(5)) receptors (pIC(50)=8.05+/xe2x88x920.03 and 8.56+/xe2x88x920.12, respectively). SRIF (0.1-1000 nM) was able to inhibit the bFGF-stimulated re-growth (pIC(50)=7.98+/xe2x88x9224 and 8.50+/xe2x88x920.12, respectively). (Alderton F et al, British Journal of Pharmacology, 124(2): 323-330, 1998).
Substance P (SP) was analyzed in rat brain endothelium cultures after cytokine stimulation. SP secretion was found after stimulation with high doses of interleukin-l beta (IL-1 beta) and tumor necrosis factor alpah (TNF-alpha). Under cytokine stimulation, part of SP was bound to brain endothelial cell surface, suggesting the existence of an autocrine network for this neuropeptide. SP regulates cellular processes in the CNS, placenta and vasculature include permeability, inflammation, mitogenesis and transformation. Increased SPR mRNA level in response to E-2 were linearly related to increased [H-3]SP binding to the SPR (Villablanca A C et al, Molecular and Cellular Endocrinology, 135(2):109-117, 1997).
The present invention provides pharmaceutical compositions for treating cancer angiogenesis and cancer metastasis. The invention provides a method of treating angiogenesis, cancer and cancer metastasis employing a pharmaceutically effective dosage of a combination of peptides or individual peptides. It is an object of this invention that a combination of peptides used is known as MuJ-7. Individual constituent peptides of MuJ-7 and pharmaceutically acceptable additives can be used. The present invention provides a pharmaceutical composition useful for killing or inhibiting multiplication of tumor cells as well as cancer cells. The pharmaceutical composition may also be useful in preventing, inhibiting, or modulating the hypersecretion of VIP, somatostain, bombesin, Substance P, or a combination of VIP, somatostatin, bombesin, or Substance P.
The composition may suitably comprise, consist of, or consist essentially of a therapeutically effective combination of peptide analogs of somatostatin, VIP, bombesin, and Substance P. The peptide analogs are described in more detail below, but constituents functionally interchangeable with those specifically described may also be employed in the claimed pharmaceutical composition. More particularly, the pharmaceutical composition may suitably comprise, consist of, or consist essentially of an analog of somatostatin and at least four peptides selected from the group consisting of a first analog of VIP, a second analog of VIP, a third analog of VIP, analog of somatostatin another analog of somatostatin, an analog of bombesin, and an analog of Substance P. More particularly, the composition may suitably comprise, consist of, or consist essentially of a therapeutically effective combination of peptide SOM2 (an analog of somatostatin) and at least four of the following peptides: VIP1 (a VIP anatagonist), VIP2 (a VIP) receptor binding inhibitor), VIP3 (a VIP receptor antagonist), SOM1 (a somatostatin analog (also abbreviated xe2x80x9cCTOP.xe2x80x9d which is derived from the first letters of the following four amino acids: Cys2, Tyr, Orn5, and Pen5), BOM1 (a bombesin antagonist), and SP1 (a Substance P antagonist). In a preferred embodiment, a pharmaceutically acceptable carrier, diluent, or solvent is used. The invention provides a method of treatment for humans, mammals, or other animals suffering from cancer or other tumors. The invention also provides a method of treatment for humans, mammals, or other animals suffering from hypersecretion of VIP, somatostatin, bombesin, Substance P, or a combination of VIP, somatostatin, bombesin, or Substance P. The method may suitably comprise, consist of, or consist essentially of administering a therapeutically effective dose of the pharmaceutical composition so as to prevent, inhibit, or modulate the hypersecretion of VIP, somatostatin, bombesin, Substance P, or a combination of VIP, somatostatin, bombesin, or Substance P.
In addition, the compositions may comprise, consist essentially of or consist of one or more of peptides identified below as DT-11; DT-12; DT-13; DT-14; DT-15; DT-16; DT-18; DT-19; DT-23; DT-24; DT-26; DT-27; DT-31; DT-33; DT-34; DT-62A; DT-62B; DT-71 which are peptide analogs.
We have observed that VIP (vasoactive intestinal peptide), somatostatin, substance P, and bombesin are secreted by at least some human tumor and cancer cells and that there are binding sites for these peptides on these cells. Specifically, out of a number of peptide growth regulators studied by indirect immunofluorescence, the four peptides (i.e., vasoactive intestinal peptide (VIP), somatostatin, Substance P, and bombesin) were shown to bind, to tumor cells. (Herein, the terms xe2x80x9cpeptide growth regulatorsxe2x80x9d, and xe2x80x9cpeptidesxe2x80x9d each refer to VIP, somatostatin, Substance P, and bombesin). It may be that there is an autocrine mechanism for cell proliferation where the peptides are secreted by tumor cells and transduce a signal through specific receptors on the same cell type leading to cell proliferation. As will be described in more detail below, the effects of the analogs of somatostatin, VIP, bombesin, Substance P on the tumor cell growth and survival were studied using different assay systems. The amino-acid sequences of the seven analogs (VIP1, VIP2, VIP3, SOM1, SOM1, BOM2 and SP1) are disclosed in U.S. application Ser. No. 08/727,679.
As will be explained in more detail below, the combination of these seven analogs is known as MuJ-7. The analogs were synthesized manually and using a conventional peptide synthesizer. An example of a combination within the scope of the invention comprises SOM2, VIP1, VIP2, VIP3, SOM1, BOM1, and SP1. A combination hereinafter referred to as MuJ-7, was prepared using the following seven peptide analogs: (1) VIP1, (the VIP antagonist) having a molecular weight of approximately 3464.9 and a concentration of approximately 10xe2x88x927 M; (2) VIP2. (the receptor binding inhibitor) having a molecular weight of approximately 1027.55 and a concentration of approximately 10xe2x88x928M; (3) VIP3, (the VIP receptor antagonist) having a molecular weight of approximately 3342.09 and a concentration of approximately 10xe2x88x928M; (4) SOM1 (the somatostatin analog (CTOP) having a molecular weight of approximately 1061.59 and a concentration of approximately 10xe2x88x929M; (5) SOM2, (the analog of somatostatin) having a molecular weight of approximately 1637.0 and a concentration of approximately 10xe2x88x928M; (6) BOM1 (the bombesin antagonist) having a molecular weight of approximately 983.55 and a concentration of approximately 10xe2x88x928M; and (7) SP1 (the Substance P antagonist) having a molecular weight of approximately 1515.83 and a concentration of approximately 10xe2x88x928M. The preceding sentence sets forth the preferred concentrations of the seven analogs comprising MuJ-expected that MuJ-7 would be effective if the concentration of each of the seven analogs ranged from approximately 10xe2x88x926M to approximately 10xe2x88x9212M.
MuJ-7 may be prepared in the following way. A stock solution of each of the seven peptide analogs is prepared with a pH of approximately 7.0 to approximately 7.4. Although sterile phosphate buffered saline was used to prepare the stock solutions for the testing described below, other diluents may be used such as RPMI 1649, buffered saline, isotonic NaCl, Ringer""s solution, water (for injection) distilled water, polyethylene glycol (near or in water), 2% Tween in water, dimethylsulfoxide to 50% in water, propylene glycol (neat or in water), balanced salt solution, glycerol, and other conventional fluids that are suitable for intravenous administration to obtain a pH in the range of approximately 7.0 to approximately 7.4 for each stock solution, the pH can be adjusted by using 1 N HCl for lowering the pH or 1 N NaOH for raising the pH, although the conventional agents for adjusting the pH can be used, the concentration of the peptide analog in each stock solution is approximately 10xe2x88x923M.
Aliquots of the seven peptides analogs are mixed together such that the MuJ-7 formulation contained approximately equal weights of each of the seven peptide analogs. In MuJ-7, approximately, the concentration of VIP1, is 10xe2x88x927M; the concentration of VIP2 is 10xe2x88x928M; the concentration of VIP3 is 10xe2x88x928M; the concentration of SOM1 is 10xe2x88x929M; the concentration of SOM2 is 10xe2x88x928M; the concentration of BOM1 is 10xe2x88x928M; and the concentration of SP1 is 10xe2x88x928M. In one exemplary embodiment, the pH of the MuJ-7 solution may range from about 7.0 to 7.4. To obtain a pH in this range, the pH can be adjusted by using 1 N NCl for lowering the pH or 1 N NaOH for raising the pH, although other conventional agents for adjusting the pH can be used.
In addition, a number of novel peptides also exhibited an antiangiogenic effect. These peptides are:
MuJ-7 was tested against primary tumor cells of human colon adenocarcinoma, and each of the peptide analogs comprising MuJ-7 was tested individually against human colon adenocarcinoma cells. A three day MTT cytotoxicity assay was performed as described in U.S. patent application Ser. No. 08/727,679. The percent killing achieved by individual peptides was in the range of 54 to 79% while percent killing achieved by MuJ-7 was 94%.
Five different subcombinations of seven peptide analogs comprising MuJ-7 were tested against human colon adenocarcinoma cells. Each subcombination was tested by performing a one day MTT cytotoxicity assay as described in U.S. patent application Ser. No. 08/727,679. The percent killing achieved by the subcombinations was in the range of 64.7% to 94.9%.